title: Aminotransferase variants as probes for the role of the N-terminal region of a mature protein in mitochondrial precursor import and processing.
abstract: Of the two homologous isozymes of aspartate aminotransferase that are also nearly identical in their folded structures, only the mitochondrial form (mAAT) is synthesized as a precursor (pmAAT). After its in vitro synthesis in rabbit reticulocyte lysate, it can also be efficiently imported into isolated rat liver mitochondria, where it is processed to its native form by removal of the N-terminal presequence. The homologous cytosolic isoenzyme (cAAT) is not imported into mitochondria, even after fusion of the mitochondrial presequence from pmAAT to its N-terminal end. Substitution of the 30-residue N-terminal peptide of the mature portion of pmAAT with the corresponding sequence from the homologous, import-incompetent cytosolic isozyme (pcmAAT) does not prevent import but reduces substantially its processing in the matrix. A detectable amount of the pcmAAT chimera is found associated with the inner mitochondrial membrane. Single and double substitution mutants of Trp-5 and Trp-6 at the N-terminal end of the mature protein are imported into mitochondria with efficiency similar to that of wild type. However, replacement of Trp-5 with proline, or of both tryptophans with either alanine (W5A/W6A mutant) or valine and alanine (W5V/W6A mutant), allows import but interferes with the correct processing of the imported protein despite the presence of an intact cleavage site for the processing peptidase. Similar cleavage results were obtained using newly synthesized proteins and mitochondrial matrix extracts. These results indicate that translocation and processing for a precursor are independent events and that sequences C-terminal to the cleavage site are indeed important for the correct maturation of pmAAT in the matrix, probably because of their contribution to the conformation and flexibility of the peptide region surrounding the cleavage site required for efficient processing. The same region from the mature component of the protein may play a role in the commitment of the passenger protein to complete its translocation into the matrix.
authors.affiliation: Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City, Missouri 64110-2499, USA.
authors.forename: B
authors.initials: B
authors.lastname: Lain
authors.forename: A
authors.initials: A
authors.lastname: Yañez
authors.forename: A
authors.initials: A
authors.lastname: Iriarte
authors.forename: M
authors.initials: M
authors.lastname: Martinez-Carrion
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001219:Aspartate Aminotransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D002451:Cell Compartmentation; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000201:enzymology / Q000378:metabolism*; D015335:Molecular Probes; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D051381:Rats
chemical_list: D004792:Enzyme Precursors; D015335:Molecular Probes; D001219:Aspartate Aminotransferases